Time-of-flight mass spectrometers with orthogonal ion injection (OTOF-MS) are commonly operated with electrospray ion sources (ESI), but increasingly they are operated also with ion sources for other types of ionization, such as chemical ionization and photoionization. Because these ion sources operate at atmospheric pressure, they have become known as atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) sources. APCI and APPI extend the scope of analyzable substances to those that are less strongly polar. Electrospray ion sources are commonly coupled with liquid chromatographs such as high performance liquid chromatographs (HPLC). APCI and APPI ion sources, however, also permit the connection with gas chromatographs (GC), since the substances separated by GC can also be ionized by APCI and APPI. This possibility extends the application of OTOF mass spectrometers, but requires accurate mass determination in the lower mass range.
Accurate mass determination is required because these ion sources deliver only molecular ions, not the signal-rich mass spectra delivered by electron bombardment ion sources (EI) usually used in GC-MS work. In prior art GC-MS, substances were identified by spectrum comparisons; libraries with hundreds of thousands of EI mass spectra are available for this purpose. However, the molecular ions delivered by APCI and APPI ion sources in OTOF-MS instruments facilitate calculating the molecular formula of the substance under investigation, if the mass accuracy is good enough. The molecular formula is first step for the identification of a substance; the measurement of fragment ion spectra of selected molecular ions may then complete the identification.
APCI and APPI ion sources regularly use high temperatures (e.g., 200° C.-470° C.), in order that the solvate sheaths are removed from the analyte molecules, without which ionization could not take place at all. The heat is supplied via the spray gas, sometimes also known as the nebulization gas. If the analyte substances are not supplied by gas chromatographs, and if the analyte solution is supplied in liquid form, the hot spray gas also has to nebulize the analyte solution and to evaporate the droplets. This method of operating the APCI and APPI ion sources can also be transferred to light analyte substances, particularly those that are either weakly polar or non-polar; here too, the lower mass range requires special measures to preserve mass precision and accuracy.
An APCI ion source uses a similar housing to that of an electrospray ion source. The spray device is operated without the spray voltage; only the spray gas is used for the nebulizing and evaporation of the solution containing analyte molecules. In order to evaporate the solvent and to remove the solvate sheath from the analyte molecules, the spray or nebulizer gas is strongly heated in a heating block to temperatures of up to 470° C. The chemical ionization is produced by reactant ions that are formed, in a chain of reactions, from primary ions of molecules of the ambient gas, usually air. These primary ions are generated in a corona discharge at the tip of a suitable metal needle, to which a few thousand volts are applied. The primary ions, usually of nitrogen, react with the water molecules in the moisture contained in the ambient gas, to form complex molecules of the form Nm(H2O)nH+ or Nm(H2O)nOH−; these are capable of causing protonation or, in a negative operating mode, deprotonation, of the analyte molecules, so causing the chemical ionization.
An APPI ion source is similar to an APCI ion source, but rather than employing a corona discharge, radiation from a UV lamp is generally used for the ionization. If a UV laser is employed, we speak of atmospheric pressure laser ionization (APLI), although this is also a type of photoionization. Only the substance molecules that can absorb the UV radiation can be directly ionized in this way; these are primarily aromatic substances. By adding an aromatic mediator substance, the mediator substance can be ionized, and its ions can then serve as reactant ions to chemically ionize many types of analyte molecule. So APPI in many cases operates as indirect APCI.
Both types of ionization can advantageously be coupled with liquid chromatography, and are used when particular substance groups of low polarity cannot be ionized by electrospraying. In contrast to biopolymers such as proteins, which can easily be ionized by electrospraying, ionization is often not successful for other kinds of organic substance; and these frequently have relatively low molecular weights.
APCI and APPI can also be coupled with gas chromatography. For this purpose the gas chromatographic separating capillary is brought into the ionization chamber of the electrospray ion source, where it releases the separated analyte substances into the ionization chamber. Coupling the gas chromatography with time-of-flight mass spectrometers of high mass accuracy provides new possibilities for quickly establishing correct molecular formulas for the analyte molecules. When gas chromatography is coupled with ion trap or quadrupole mass spectrometers via electron impact ion sources, which has been the usual method up to now, the relatively low mass accuracy only permits the identification of substances through spectral comparisons; this fails in the case of unknown substances, for instance in the analysis of unknown natural products.
Coupling an OTOF mass spectrometer with a gas chromatograph has also led to the development of APLI. Here, the photon density in the beam from a UV pulse laser is exploited to ionize aromatic substances directly by multiphoton processes. For this purpose, a rectangular cross-section of the laser light pulse beam is created directly in front of the GC capillary; with each laser light pulse, the width of the beam catches a proportion of the carrier gas as it flows out, so in combination with the laser light pulse frequency (usually 100 hertz), the whole of the GC eluate is recorded. In this way, the aromatic substances in the GC eluate are detected with great efficiency and high sensitivity. Through the additional use of mixtures of aromatic mediators such as benzene, toluene or similar substances, and of non-aromatic mediators such as chloroform, it is also possible to ionize many non-aromatic substances.
In the development of OTOF mass spectrometers, improvement of the mass accuracy is crucial for determining the masses of analyte ions. The aim is to achieve a mass accuracy of better than a millionth of the mass (one ppm). Until now, however, attention has been paid to the mass range extending from about 500 up to a few thousand daltons because the main field of application of these mass spectrometers is the analysis of biopolymers, primarily proteins. Mass accuracy has until now been neglected in the range below 500 daltons; only recently has it been recognized that, in the lower mass range, the calibration curve differs significantly from the ideal calibration function, preventing accurate mass determination.
U.S. Published Patent Application 2008/0308724A1 discloses how deviations of the calibration curve from the theoretically expected curve in the lower mass range can be explained and described mathematically to a good approximation. The approximation allows the masses of the light ions in the lower mass range from about 100 to 500 daltons to be determined to within almost a millionth of the mass (1 ppm) if the coefficients of the mathematical equation for the calibration curve can be determined, by a sufficient number of reference points, precisely enough for a calibration. It is particularly advantageous if the reference points are separated from one another by the same mass difference.
In OTOF mass spectrometers, the ions from a section of a fine beam of ions are suddenly accelerated, perpendicular to their former flight direction, into the flight path, by an ion pulser. They are then reflected by a reflector at a slight angle onto the ion detector, where they are measured as a time-variable ion current that represents the time-of-flight spectrum. The accelerating voltages in the ion pulser, however, cannot be switched instantaneously, due to the capacitances of the lines and the pulser. In the best case of a critically damped switching, no overshooting and no oscillations occur and the voltage follows a transition curve whose time constant is a few nanoseconds long. Very light ions that can be accelerated quickly pass through the pulser before the full accelerating voltage has been reached. This results in deviations of the calibration curve m/z=f(t) from the ideal curve m/z=a×t2, where m/z represents the mass m per elementary charge, t the time of flight, and is a constant. As mentioned, the best results for a relatively smooth calibration curve in the lower mass range are obtained by trying to achieve the critically damped case. If the ideal non-oscillating critically damped case is not achieved precisely, then either overshooting with subsequent oscillations or slow settling will occur, resulting in even larger, sometimes irregular, deviations. Unfortunately it is not always possible to maintain the non-oscillating conditions of critical damping because, for instance, aging processes in the electronic equipment or temperature changes cause the settings to drift.
The equations for best approximation given in U.S. Published Patent Application 2008/0308724A1 can be used to calibrate the mass spectrometer in the lower mass range. It is, however, also possible to perform accurate mass determinations without the knowledge of a mathematical approximation equation, by interpolating the masses between the stored reference points of the mass scale with an n-th order polynomial curve. Usually here, the time of flight t is first squared to achieve a rough linearization with reference to the mass values m, since, apart from the deviations considered above, the masses are proportional to the square t2 of the flight time in a time-of-flight mass spectrometer. Here again it is advantageous if the reference points are separated from one another by the same mass difference.
Because the APCI, APPI and APLI ionization sources essentially deliver singly charged molecule ions, and no fragment ions, the generation of a mass spectrum with enough reference points of precisely known masses requires a calibration substance for each reference point; thus a mixture of calibration substances is needed.
Mixtures of substances that can be used to calibrate the mass scale in ESI-OTOF mass spectrometers are already known. Until now, however, these mixtures have always been intended for use in the high mass range between 500 and a few thousand daltons; there are only very few reference points in the lower mass range. For instance, in the work of S. J. Stout and A. R. daCunha, entitled “Tuning and Calibration in Thermospray Liquid Chromatography/Mass Spectrometry Using Perfluorinated Alkyl Acids and Their Ammonium Salts”, which comes closest to the invention disclosed here because of the substances used, a mixture entirely of fluorinated fatty acids and their ammonium salts is described. However, this mixture only supplies two reference points in the mass range below 500 daltons. Furthermore, the salts decompose at the high temperatures that are applied here.
U.S. Pat. No. 5,872,357 discloses a mixture of various substituted triazatriphosphorine compounds. The mass spectrum of the mixture offers reference points with uniform mass spacings of either 300 or 600 daltons, whose masses are known precisely. Unfortunately, however, this mixture again only provides two reference points in the mass range up to 500, although already a non-related substance is added that provides a further reference point in the low mass range. This mixture of calibration substances is marketed with great commercial success, but for the reasons mentioned above can only be used for accurate mass determinations in the higher mass range from about 500 daltons up to around 3000 daltons. In this higher mass range, the calibration curve can be represented precisely by approximation equations whose curves are smooth and stiff, and require only a few reference points.
The requirements for a mixture of calibration substances for the lower mass range are as follows:                the calibration substances should be thermally stable at temperatures up to 470° C.;        they should supply at least five, and preferably ten, reference points below 500 daltons;        the masses of the calibration substances should be as evenly spaced as possible;        the calibration substances should be able to form both positive and negative ions;        the calibration substances should be nontoxic;        the calibration substances should be soluble in one another; and        the solution of calibration substances should be capable of pulsed injection, and the signals from the calibration substances should rapidly diminish again, without a memory effect.If these requirements are satisfied, then this mixture of substances can be used to develop an automated calibration method, which can be run regularly prior to, or even during, analytical procedures, and can therefore compensate for drifting of the mass scale.        